The present invention relates to a method for the preparation of a polypeptide, more particularly it relates, to a method for the preparation of a polypeptide by the selective sulfation of OH-groups of Tyr in a starting material having Tyr, Ser and/or Thr residues.
Recently, polypeptides have drawn enthusiastic attention as a type of protein useful in genetic engineering. Synthesis of various types of polypeptides has been hitherto attempted. The synthetic methods hitherto proposed include the solid phase method and the liquid phase method. The former method is not suitable as an industrial method of preparation because of the low purity of the resultant peptide and the many difficulties encountered in the final stage of purification. The liquid phase method, on the other hand, is further classified into the stepwise method and the fragment condensation method, the latter of which is currently used to avoid the difficulty in the purification of the product unavoidable in the former method as in the solid phase method.
The fragment condensation method has several advantages as compared to the step-wise method. Namely, the synthesis can be formed fragment by fragment and the loss of product can be decreased. In addition, the final product is easily formed and has a high purity. However, the fragment condensation method is accompanied by the possibility that the amino acid residue of the carbon terminal is susceptible to racemization in the condensation reaction. This racemization detracts from the advantages of the fragment condensation method. It can be partially solved by appropriate selection of the combination of the fragments as is known in the art.
Success has hitherto not been reported in the attempts for the total synthesis of human cholecystokinin, referred to as hCCK-33 hereinbelow, as a polypeptide by the fragment condensation method. The reason for the unsuccessful outcome is because a reagent is not available for the selective sulfation of the Tyr residues alone in the presence of amino acid residues such as Ser and Thr when successive azide condensation reaction is attempted on a plural number of fragements having protective groups and that the sulfation by a reagent occurs not on the desired Tyr-OH but preferentially on the Ser-OH or Thr-OH.